Analytica Chimica Acta 555 (2006) 242–245

Determination ofl-ascorbic acid in wines by direct injectionliquid chromatography using a polymeric column

Paulo Lopes, Jessica Drinkine, Cedric Saucier∗, Yves GloriesFaculte d’enologie de Bordeaux, Universite Victor Segalen Bordeaux2 UMR 1219 INRA 351 Cours de la liberation,

33405 Talence Cedex, France

Received 19 May 2005; received in revised form 29 August 2005; accepted 2 September 2005Available online 10 October 2005

Abstract

A rapid method for the identification and quantification ofl-ascorbic acid in wines by direct injection liquid chromatography equipped with aUV detection was developed. The levels of ascorbic acid were determined using a polymeric PLRP-S 100 A (5�m) column (150 mm× 4.6 mm)with a mobile water/trifluoroacetic acid (99/1, v/v) phase. The method is rapid (less than 5 min) and sensitive (LOQ of 5 mg L−1). The calibrationcurve of ascorbic acid was linear (r = 0.999) over a concentration range between 1 and 200 mg L−1. Repeatability was less than 2.5% and ther©

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eywords: Liquid chromatography;l-ascorbic acid; Wine

. Introduction

Ascorbic acid, commonly known as Vitamin C, is a powerfulatural antioxidant present in a wide range of foods and bever-ges. It is present in grapes (50 mg L−1), but in wine productionisappears during fermentation[1]. However, it is purposelydded to wines, particularly to white wines during winemaking,

o prevent oxidation and to prolong the shelf-life of the commer-ial wines[2–4].

The ability of ascorbic acid to scavenge molecular oxygens the basis for its addition during winemaking, particularly justefore bottling[1,2]. Its antioxidant capacity prevents the oxida-

ive spoilage of white wines, which can have a negative impactn the wine’s flavor and color. For this reason, ascorbic acid cane used as a marker of white wine’s bottle ageing[5].

In a first step, the aerobic oxidation of ascorbic acid producesoth dehydroascorbic acid and hydrogen peroxide. However,

here is evidence that other products of ascorbic acid breakdownre responsible for the high levels of browning in some whiteines[3,4,6].

Various methods have been described for the determinatascorbic acid in foods and beverages. These include calori[7], spectrophotometry[8–10], voltammetry[11,12], thin-layerchromatography[13] fluorimetry[14,15]and enzymatic assa[16,17]. However, some of these methods present draw-bsuch as, low sensitivity and selectivity and/or difficult sampreparation.

The liquid chromatography (LC) has been the mosttechnique, based on reversed-phase C18[18–23], bonded-phasNH2 [24], anion-exchange[25], ion-pair reversed-phase C[26] and electrochemical detection[27,28] and UV detection[18,19,23,26]. However, very few methods are capable for didetermination of ascorbic acid in wines.

This paper describes a rapid method for the identificationquantification of ascorbic acid by direct LC injection of winusing a polymeric column. The application of this methosome white and red wines is briefly discussed.

2. Materials and methods

2.1. Reagents and materials

∗ Corresponding author. Tel.: +33 5 40 00 64 87; fax: +33 5 40 00 646.E-mail address: saucier@oenologie.u-bordeaux2.fr (C. Saucier).

Deionized water was purified with a Milli-Q water system(Millipore, Bedford, USA) prior to use. Acetonitrile (HPLCgrade) was obtained from Fisher Chemicals (Elancourt, France)

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.09.005

P. Lopes et al. / Analytica Chimica Acta 555 (2006) 242–245 243

and Trifluoroacetic acid 99%, acetic acid 99–100%, formicacid 99–100% andO-phosphoric 85% acid were obtainedfrom Prolabo-VWR (Fontenay S/Bois, France).l-ascorbic acidwas purchased from Sigma–Aldrich (Saint Quentin Fallavier,France). The syringe membranes filters (GHP Acrodisc®

25 mm, 0.45�m) were purchased from Pall Life Sciences (AnnArbor, MI, USA). Commercially available wines were used.

2.2. Standard and sample preparation

A model wine solution was prepared containing 12% (v/v)ethanol, 5 g L−1tartaric acid and adjusted to pH 3.2 with 1Nsodium hydroxide. A 20 mg ofl-ascorbic acid was dissolvedin 100 mL of model wine solution and diluted to the desiredconcentration (1–200 mg L−1) with the same solution.

All the samples (white and red) were filtered through mem-branes filters (GHP Acrodisc® 25 mm, 0.45�m), which did notretain the ascorbic acid. The filtrate (2 mL) was directly injectedinto the LC system.

Standards and samples were prepared daily and stored in2 mL aliquots at 4◦C in a refrigerator in total darkness priorto injection[27].

2.3. Apparatus and conditions

2.3.1. Apparatusa

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diode array detector allowed to detect an absorption maximumof 243 nm for our conditions in the UV spectra.The externalstandard method was used to quantify the ascorbic acid con-centration at 243 nm. Calibration curves (standard area versusconcentration in mg L−1) were performed in triplicate with aconcentration range of 1–200 mg L−1 corresponding to the rec-ommended quantity for addition to wines.

The linearity of the standard curves were expressed as a corre-lation coefficient (r) by plotting the integrated peak area againstthe concentration of the standard (mg L−1). The repeatabilitywas obtained for the retention time and with the determina-tion of the ascorbic acid concentration. The data were obtainedfrom 12 replicate injections a concentration of 25 mg L−1 ofthe wine model solution, white (Sauvignon) and red wine(Merlot).

The limit of detection (LOD) was determined from repeatedinjections of diluted solutions of ascorbic acid in a model winesolution, at a signal-to-noise ratio (S/N) of 3. The limit of quan-tification (LOQ) was obtained at an S/N of 10. A specific amountof ascorbic acid was added to a white (Sauvignon) and a red wine(Merlot) for the estimation of the recovery with the standardaddition method.

3. Results and discussion

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LC/DAD analysis were performed by means of a Beckmold System (Roissy Charles-de-Gaulle, France) using a m

njector, a 126 pump module and a 168 diode array detell systems were operated using 32 Karat 5.0 software. UVere recorded from 200 to 900 nm and peaks were measu43 nm (maximum absorption of ascorbic acid).

.3.2. ColumnsThe separation was performed on a PLRP-S 100A (5 �m)

olumn (150 mm× 4.6 mm) (Polymer Laboratories, ShropshK).Others columns tested were Xterra RP 18 (3.5�m) column

100 mm× 4.6 mm) (Waters, Milford, Massachusetts, USirchromn-EZ 300 A (3�m) column (100 mm× 4.6 mm)

Zirchromn, Anoka, MN, USA), AtlantisTM dC18 (3�m)olumn (100 mm× 4.6 mm) (Waters, Milford, MassachuseSA) and Beckman ODS ultrasphere (5�m) column

250 mm× 4.6 mm) (Beckman Coulter, Fullerton, CA, USA

.3.3. Mobile phaseElution conditions were as follow: flow rate 1 mL min−1

t room temperature, 20�L sample loop; Solvent Aater/trifluoroacetic acid (99:1, v/v); solvent B, aceto

rile/solvent A (80:20, v/v). The gradient elution profile ws the following: 0–5 min 100% A, 5–6 min 100% B, 6–10 m00% B, 10–11 min 100% A, 11–12 min 100% A.

.4. LC procedure

The ascorbic acid was identified by comparing its retenime with external standards. The use of LC with a ph

nualor.isd at

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The purpose of this investigation was to develop a rLC method for the determination of ascorbic acid in redwhite wines, whilst keeping the sample preparation to a mmum in order to use this method for routine analysis withcosts.

3.1. Column and mobile phase

For the development of this method, we used diffecolumns and mobile phases considering the hydrophobicitpH resistance of the stationary phases. Preliminary trialsmade by comparing the different columns. Different mobphases were also tested, particularly with solvent A in conation with different acids (acetic acid, formic acid, phosphacid).

All the columns showed good resolution and selectivityascorbic acid detection in model wine solution after the omization of the mobile phases. However, when the identition of ascorbic acid was performed in wines (added ascacid), all the columns displayed poor resolution and seleity with the respective optimized mobiles phases. To obtagood separation of ascorbic acid it was necessary to incthe acidity of the mobiles phases by replacing the formicwith trifluoroacetic acid. A mobile phase of water/trifluoroac(99/1, v/v) acid was chosen (pH 1.4). At this very lowvalue only the PLRP-S 100A (5 �m) column could work efficiently and was resistant to this acidic conditions. All othcolumns were not recommended to work at this pH. Tcolumns can perform an acceptable resolution and selecof ascorbic acid although the column shelf-life would likelyreduced.

244 P. Lopes et al. / Analytica Chimica Acta 555 (2006) 242–245

3.2. Peak identification

A good peak shape and a suitable retention time (2.3 min) forascorbic acid was obtained. The dehydroascorbic acid was notquantified because it is weakly detected in UV[19,23]. Otherspeaks were also detected in white and red wines, which made theidentification and quantification of the low amounts of ascorbicacid less certain (Fig. 1). It was noted that an unknown peakwas located on the tail of ascorbic acid. This area correspondedto almost 1 mg L−1 of ascorbic acid, which made it difficult toquantify the amounts close to the limit of quantification (LOQ)of ascorbic acid in wines. Taking into account this matrix effect,we propose a limit of detection (LOD) for the ascorbic acidequal or slightly higher than LOQ obtained with the model winesolution (see Section3.3.3).

3.3. Method validation

3.3.1. LinearityBased on the peak area obtained (y) against the stan-

dard concentration (x), the following equation was found[y = 45094x + 13117] (that intercept fairly close to zero).

The correlation coefficient,r = 0.999 was highly significant(p < 0.01) over a range of concentrations (1–200 mg L−1).

3time

w was1 forw ding

to the criteria determined by Horwitz for the intra-laboratoryanalysis[29].

3.3.3. SensitivityThe LOD in model wine solution was 360�g L−1, while the

LOQ was 1 mg L−1. Because of the matrix effect as describedearlier (Section3.2) the LOD in wine solution was set up to1 mg L−1, while the LOQ was set up to 5 mg L−1.

3.3.4. RecoveryAscorbic acid recovery was over 90% for the model wine

solution and wines (Table 1).

3.4. Method application

The proposed method was applied to the determination ofascorbic acid in some white and red wines. Ascorbic acid wasnot detected in the analyzed red wine samples. These resultscould be considered normal as ascorbic acid disappears duringthe fermentation. Additionally, the addition of ascorbic acid isnot considered to be acceptable in red wines.

The amounts of ascorbic acid measured in different whitewines is summarized inTable 2. The concentrations varied anddepended on the winemaking practices of each winery. Nev-ertheless, these amounts are in agreement with the limits ofa

umnp tables surea uid

F odel )(

.3.2. RepeatabilityThe relative standard deviation (R.S.D.) of the retention

as lower than 0.8%, while the R.S.D. of the concentration.6% (model wine solution), 2.2% for red wine and 1.5%hite wine (Table 1). These values are acceptable accor

ig. 1. Chromatographic analysis (λ = 243 nm) of ascorbic acid (aa) in a m

30 mg L−1).

scorbic acid recommended by the O.I.V. (<250 mg L−1) [30].Under our conditions, the use of a polymeric PLRP-S col

roduced a fast separation of ascorbic acid with an accepensitivity and selectivity. This method is capable to meascorbic acid concentrations in wine by direct injection liq

wine solution (A) (25 mg L−1), white wine (B) (20 mg L−1) and a red wine (C

P. Lopes et al. / Analytica Chimica Acta 555 (2006) 242–245 245

Table 1Average and repeatability study for the retention time, concentration and recovery of ascorbic acid at 25 mg L−1 (n = 12)

tr (min) Repeatability (%) Concentration (mg L−1) Repeatability (%) Recovery (%)

Model wine solution 2.3 0.55 24.9 1.6 100White wine 2.4 0.54 24.1 1.5 96.1Red wine 2.5 0.58 22.9 2.2 91.9

Table 2Ascorbic acid contents (mg L−1) in white and red wines

Wines A.O.C Measured value(mg L−1)

White winesSauvignon/Semillion

2003Cote de Duras 35

Sauvignon/Semillion2003

Cote de Duras 89

Sauvignon 2004 Cote de Duras 78Sauvignon 2004 Pessac-Leognan 104Sauvignon 2002 Pessac-Leognan 45Sauvignon 2002 Pessac-Leognan 23Sauvignon 2004 Graves 110Sauvignon 2004 Graves 87

Red winesMerlot 2002 Saint Emilion n.d.Merlot 2002 Saint Emilion n.d.Carbenet

sauvignon/Carbenetfranc 2004

Saint Emilion n.d.

Carbenet sauvignon2003

Medoc n.d.

Carbenet sauvignon2003

Medoc n.d.

chromatography with almost no sample preparation. The pro-cedure uses only UV detection, the total analysis time being12 min including washing and re-equilibration of the system.The method presented here is then suitable for routine analysin laboratories equipped with a UV-LC.

4. Conclusion

The use of a polymeric PLRP-S column allows a good separation of ascorbic acid by direct injection liquid chromatography.The method proposed here is simple and rapid with a shortewashing re-equilibration times. This method can be applicablefor routine analysis of ascorbic acid in wine, especially in whitewines where the addition of this compound before bottling iscommon practice. The level of this compound can also be useto evaluate the oxidation state of white wines.

Acknowledgments

The authors whish to thank Amorim France (Eysines, Franceand ANRT [Association Nationale pour la Recherche Tech-nologique (Paris, Cifre Grant No. 097/2004) for their financial

support in this research. The authors also acknowledge AdriaanOelofse for revising the manuscript.

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